Rolling Circle Amplification

Abstract

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.

Recent developments in multiplexing techniques for immunohistochemistry

“…Rolling circle amplification (RCA, Figure 4D) is a powerful DNA replication based tool that offers high sensitivity and enables subcellular level analysis within tissue without increasing background noise [1,74]. The RCA method involves the conjugation of the 5′ end of an oligonucleotide (OLN) primer to an antibody, and a circular single-strand DNA template is annealed to the OLN primer.…”

“…In the presence of DNA polymerase and nucleotides, the primer extends at its 3′ end to create multiple copies of the circular DNA [75,76]. The resulting amplicon is labeled through hybridization with fluorescently tagged OLNs [74–76]. RCA differs from the polymerase chain reaction in that the DNA replication is carried out at a specific antigen site, and in isothermal conditions, linear or geometric kinetics can be used to amplify round OLN probes [74,75].…”

“…The resulting amplicon is labeled through hybridization with fluorescently tagged OLNs [74–76]. RCA differs from the polymerase chain reaction in that the DNA replication is carried out at a specific antigen site, and in isothermal conditions, linear or geometric kinetics can be used to amplify round OLN probes [74,75]. The exponential and linear mode RCA amplification is respectively able to generate a 109- and 105 fold signal amplification in a short period of time[74].…”

“…Detection with 1:25,600 antibody dilution (E) Polymer-Based signal amplification involves binding a polymer backbone that is conjugated with multiple antibodies and enzymes for higher contrast visualization. Permissions were obtained for microscopy images of stained cells and tissues (enclosed in shaded boxes) [86], [72], [87],[74], [88]. …”

“…Microscopy images of stained cells and tissues (enclosed in shaded boxes) are reproduced with kind permission from Nature Publishing Group (A) [86], Springer Science and Business Media (B) [87], Methods, 18(4) Soontornniyomkij V, Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy, 459–464, Copyright 1999, from Elsevier (C) [72], the American Journal of Pathology, 159(1), Gusev Y, Sparkowski J, Raghunathan A et al Rolling Circle Amplification: A New Approach to Increase Sensitivity for Immunohistochemistry and Flow Cytometry, 63–69, Copyright 2001, with permission from Elsevier (D) [74], and Wolters Kluwer Health (E) [88]. …”